Protein Adsorption at Interfaces Detected by Second Harmonic Generation

Abstract

We show that second harmonic (SH) spectroscopy, an intrinsically surface-selective technique, can be used to monitor protein (cytochrome c) adsorption to silica surfaces and negatively charged supported phospholipid bilayers. The origin of the SH signal is due to the effect of the adsorbed protein on the water molecules polarized near the charged interface. Although the protein does not contribute its own SH signal, its binding to the glass surface or the membrane reduces the polarization of the interfacial water molecules and this effect is proportional to the adsorbed protein concentration and can be monitored with high precision, in real time. The free energy of adsorption (∆G ads ) -11.8 kcal/mol) of cytochrome c to glass was determined from an adsorption isotherm measurement of the SH signal as a function of the bulk protein concentration. A detection sensitivity of 0.1 pmol/cm 2 of adsorbed cytochrome c protein is readily achieved.

Reorientation of the helix of the tryptophan-rich gp41W peptide from HIV-1 at interfaces

“…However, it has been shown that a decrease of the overall SHG intensity from the interface can also be observed as for example in the case of cytochrome c if the contribution of a static dc-field is taken into account. 38 In the present study, the SHG increase appeared on a much faster time scale than the corresponding surface pressure (20 min versus 2 h respectively after gp41W injection). The surface pressure delay likely underlines already internal re-arrangements within the gp41W upon adsorption as compared to the bulk aqueous phase conformation.…”

Vibrational Spectroscopic Investigation of the Phase Diagram of a Biomimetic Lipid Monolayer

“…Second-order nonlinear spectroscopies with their intrinsic surface sensitivity [3] have provided important molecular-level insights into biological surface systems [4]. In particular, VSFG [5] has been used to probe DPPC phospholipid layers at the liquid-liquid interface as well as the kinetics of bilayer formation from vesicle rupture [6] by monitoring C -H stretch vibrations of both CH 2 and terminal CH 3 groups in the alkyl chain.…”

Environment-Induced Surface Dynamics of a Biomimetic Ionomer Studied Using in Situ Second Harmonic Generation

“…This value is comparable to or less than that used in other SHG or SFG studies involving polymers. 18,20,26 Additionally, it is important that the sample is not heated by the laser during the experiment as this would affect the molecular dynamics. The lack of absorbance bands at the laser wavelength is a benefit in this respect, and the laser power levels used in this study are less than levels that have been verified to prevent heating of thin polymer samples.…”